Lysosomal reduced thiols are essential for mouse embryonic development

Abstract

While it has been appreciated for decades that lysosomes can import cysteine, its significance for whole-organism physiology has remained uncertain. Recent work identified MFSD12 as a transmembrane protein required for cysteine import into lysosomes (and melanosomes), enabling genetic interrogation of this pathway. Here, we show that Mfsd12 knockout mice die between embryonic days 10.5 and 12.5, indicating that MFSD12 is essential for organogenesis. Mfsd12 loss results in the expression of genes involved in cellular stress and thiol metabolism and likely disproportionately affects the erythroid, myeloid, and neuronal lineages. Within lysosomes, imported cysteine is largely oxidized to cystine, which is exported to the cytosol by the cystinosin (CTNS) transporter. However, unlike Mfsd12, loss of Ctns is compatible with viability, suggesting that the essential role of MFSD12 lies not in supplying cystine to the cytosol, but in providing reduced cysteine within the lysosomal lumen. Supporting this model, maternal treatment with cysteamine-a lysosome-penetrant thiol-rescued the development of Mfsd12 knockout embryos, yielding viable adult offspring. These findings establish lysosomal thiol import as a critical metabolic pathway and provide genetic tools to further clarify its physiological and biochemical roles.

Keywords: MFSD12; cysteine; lysosome; redox.

Fig. 1.

Loss of Mfsd12 in mice results in completely penetrant lethality. (A) Diagram of murine Mfsd12 gene structure and the location of the CRISPR-induced Mfsd12 mutation. (B) Nucleotide and amino acid resolution diagram of Mfsd12^em1Hadel mutation, showing the predicted amino acid sequence of the Mfsd12 knockout allele. This includes an annotation of transmembrane domain IV from Uniprot Q3U481. Note the loss of hydrophobic amino acids (I, V, F etc.) (C) qRT-PCR analysis of Mfsd12 expression in the kidney and spleen comparing Mfsd12^+/+ and Mfsd12^+/− matched, male littermate pairs. Data are shown as mean ± SEM, with significance determined by unpaired, two-tailed Student’s t test (*P < 0.05, **P < 0.01, n = 3 pairs of mice). (D) Offspring genotypes from 12 monogamous Mfsd12^+/− x Mfsd12^+/− crosses. The P-values are from a Chi-squared test for Mendelian ratios (n = 123 mice). (E) Representative photographs of 4-wk-old Mfsd12^+/+ and Mfsd12^+/− littermates show no obvious differences in body size or coat pigmentation.

Fig. 2.

Mfsd12 knockout induced embryonic lethality manifests 10.5 to 12.5 d after fertilization. (A and B) Photographs of littermate embryos staged at mE11.0 and mE12.2 illustrate the progressive defects in Mfsd12 knockout embryos. All Mfsd12 knockout embryos shown were classified as “abnormal” based on size and morphology. Precise embryo staging was done by averaging limb staging data from eMOSS tool for “normal embryos” per litter (22, 23). (C and D) Tables showing the number of embryos isolated at indicated days post-fertilization and their genotypes. Embryos that were not visibly different from wild type are included in Fig. 2C, while those that were deemed grossly abnormal based on morphology or small size are in Fig. 2D. The P-values are from a Chi-squared test for Mendelian ratios calculated for “Normal Embryos” (n = 61, 42, 40 embryos). (E) Volcano plot of differential gene expression analysis comparing the protein-coding, autosomal transcriptomes of Mfsd12 knockout and wild-type embryos (n = 4 per genotype). (F and G) GSEA of Mfsd12 knockout versus wild-type embryos using the Hallmark pathways collection (via MSigDB) and the Mouse Cell Atlas (24, 25). The top 10 gene categories by FDR are plotted for each analysis. The legend is shared between panels. (H) ChEA3 transcription enrichment using differentially expressed genes from Mfsd12 knockout versus wild-type embryos. The top 10 transcription factors identified by mean rank are plotted.

Fig. 3.

Knockout of Ctns does not phenocopy that of Mfsd12 with respect to embryonic lethality. (A) Diagram of Ctns gene structure and the location of the CRISPR-induced Ctns mutation. The diagram is oriented in the antisense direction. (B) Nucleotide and amino acid resolution diagram of Ctns^em1Hadel mutation. (C) qRT-PCR analysis of Ctns expression comparing three Ctns^+/+ and Ctns^−/− matched male littermate pairs. Data are shown as mean ± SEM, with significance determined by unpaired, two-tailed Student’s t test (****P < 0.0001, n = 3 pairs of mice). (D) Number and frequency from 4 monogamous Ctns^+/− x Ctns^+/− crosses. The P-values are from a Chi-squared test for Mendelian ratios (n = 89 mice). (E) Photograph of 4-wk-old Ctns^+/+ versus Ctns^−/− littermates showing no obvious coat color differences.

Fig. 4.

Lysosomal thiol supplementation with cysteamine rescues Mfsd12 knockout animals to live birth. (A) Genotype ratios of neonatal mice (found both deceased and alive) identified on the day they were born (P0) from mothers treated with control water (0.3% sucrose, pH 6.0), cysteine-supplemented water (8 mM cysteine, 0.3% sucrose, pH 6.0) or cysteamine-supplemented water (8 mM cysteamine, 0.3% sucrose, pH 6.0). The P-values are from a Chi-square test for Mendelian ratios (n = 58, 35, 99 neonates) (B) Photograph of anesthetized neonates including live and deceased Mfsd12^−/− neonates, typical of cysteamine rescue. For each treatment category, littermates are shown. Photographs of control and cysteamine-treated mice were obtained on different days. (C) Neonate forepaws (ventral right and left paws) from the same treatment as (A and B). The paws feature digits parallel to the midline and the presence of distal phalanges, features of development associated with progression past ~E16 (23). (D and E) Schematic and results from an experiment testing the viability of Mfsd12 knockout mice after withdrawal of cysteamine at postnatal day 7. The P-values are from a Chi-square test for Mendelian ratios (n = 34 neonates). (F) Photograph of 3-mo old adult female littermates which were withdrawn from cysteamine 1-wk postnatally. The gray coat color of the Mfsd12^−/− animal is consistent with other reports of Mfsd12 deficiency on an agouti background (47, 49). (G) Model of lysosomal cysteine and cystine metabolism in mouse embryonic development, highlighting the importance of luminal thiols like cysteine as reducing agents over cystine storage and release to the cytosol.